| Human papilloma virus (HPV) infection was essential for genesis ofcervical cancers. After infection, E6and E7gene of HPV were integratedto genome of cervical epithelium. Continued expression of transformingoncoprotein E6not only drives the neoplastic progression in cervicalepithelium but also plays important role in maintaining malignantphenotype of cervical cancer cells. HPV-DNA could be founded in90%ofcervical cancer,60%of it was type16and18. Since the integrated HPVtransforming genes have no appreciable sequence homology to humangenome, they become attractive targets for biotherapy. Blocking of HPV16E6could not only give chance to discover more detials of cancerization ofcervical epithelium, but also has potential application value in biotherapyof cervical cancer.Antisensenucleic acids and targeting ribozyme wereusually applied to block E6gene expression to explore mechanism ofcervical cancinogenesis. However, their blocking efficiency was limited,and it was difficult for them to reach cells in vivo. RNA interfering hasbeen utilized to elucidate gene function employing double-strand RNA thatis capable of degrading mRNA in a sequence-specific manner. Smallhairpin RNA (shRNA), which was transcripted from expression sequencein cells, could silence target gene function over7days. Further more,shRNA was very convenient for working in vivo. Lentivirus wascharacterized with high infection ability and faint stimulation to immunesystem. Thus, lentivirus would be commendable carrier for shRNA.There was, however, not any reference till now about that shRNA carried by lentivirus could block E6expression. In the hope of developing agene-specific therapy for HPV related cancer, we designed three expressionsequences of shRNA target to HPV16E6（E6-shRNA）, and cloned thesequences to lentivirus work vector PLL3.7. The recombined vectors wereidentified by restriction enzyme analysis and sequencing. Lentivirus wasproduced after PLL3.7and three package plasmids were cotransfected to293FT cells with the help of lipofectamine2000. Cervical cancer Caskicells were infected by recombined lentivirus. We examined the effects ofE6-shRNA on the expression of HPV16E6and its related proteins P53andP21. Proliferative ability of cancer cells was determined by counting theamount of cencer cells each day after interfering. The invasion character ofCaski cells were also analyzed after infection employing transwell kit. Theresults were as follow.1. The lentivirus was produced successfully by293FT cells with titerof5106TU/ml.2. When Caski cells were infected by recombined lentivirus, the levelsof mRNA encoding HPV16E6in cells was reduced by80%, andexpression of E6protein was reduced by65%. In addition, the amount ofP53and P21protein in cells was increased significantly.3.7days after shRNA-lentivirus infection, the proliferative ability ofCaski cells was found to have been decreased, the amount of Caski cells inthe test groop was only as45%as that in the control group. Examined bytranswell test, invasion character of Caski cells was found to be reduced,the number of cells passing through mini cellular membrane in the testgroup was decreased by65%compared with the control.As limited by package system, the titer of most recombined retroviruscould only reach105TU/ml. We demonstrated that lentivirus packaged by293FT could reach a primary titer of above106TU/ml, which was different from general retrovirus. The appearance and proliferative ability of Caskicells had no change after nonsense lentivirus infection. These suggest thatno additional cytotoxicity happened if augmented lentivirus was applied toCaski cells; however, this could increase the ratio of infected Caski cells.We concluded that lentivirus was of very valuable application in RNAi dueto its high titer and low cytotoxicity; it was perfected carrier for conductinggene to cervical cancer cells.One important transforming course in cell was HPV16E6proteinbinding with p53and resulting in the ubiquitin mediated degradation of p53.The tumor suppressor gene p53serves within the checkpoint circuits thatregulate cell division. That means to ensure the completion of cycledependent events in the cell division cycle and provide more time for DNArepair before DNA replication and mitosis. P53could activate WAF gene,and result in accumulation of its product P21protein in cell. P21could bindwith and suppress kinase of CDK family, and this could result in stoppingof cell cycle. Inactivation of P53, which would lead to dysfunction ofproliferation related factors such as P21, PCNA, Cyclins and Cdks,probably plays a crucial role in the development of cervical cancer. If thishappened, damage of DNA would accumulate and cancerization wouldoccur. We demonstrated that lentivirus could integrate shRNA expressionsequence to Caski cells genome after infection and produce E6-shRNAafter transcription. ShRNA could reduce65%of E6protein accompaniedby an increasing of P53and P21protein expression. It suggested that ourrecombined lentivirus could suppress E6expression efficiently as long asone month. It offered a good tool for us to discuss downstreamimmortalization mechanisms of E6.The continued expression of E6plays an important role in maintainingmalignant phenotype of cervical cancer cells. Our investigations in cellular level show that a great drop of proliferative ability would followsuppression of E6expression in Caski cells. The number of cells passingthrough mini cellular membrane in the test group were also decreased.These results suggested that malignant phenotype of Caski cells could bechanged by suppression of E6expression. The shRNA mediated bylentivirus may have potential application value in biotherapy of cervicalcancerConclusions: We had successfully constructed shRNA-lentiviruswhich could effectively suppress E6expression in cells, this provided asuitable tool for exploring E6transforming downstream mechanism. It wasproved that shRNA could change malignant phenotype of Caski cells incyto level. It was demonstrated primarily that lentivirus based shRNA hadattractive potential in cervical cancer biotherapy in vivo. |
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